PROJECT SUMMARY/ABSTRACT The high stability of local structure for RNA and DNA has profound and widespread impacts on life. For structured RNAs, high local stability enhances the ability of RNAs to fold by progressive formation of modules, but it also increases the odds and the consequences of misfolding. For cellular processes involving RNA or DNA, protein enzymes are required to transiently disrupt nucleic acid structure. This framework provides the overarching theme of the research of my group. In the area of RNA folding, we are using model RNAs derived from a group I intron to test whether RNA folding kinetics can be understood and ultimately predicted by understanding the properties of the modular tertiary contacts and junctions that underlie the folding of these RNAs. In the area of protein-mediated changes in nucleic acid structure, our current research interests encompass three projects. (1) DEAD-box helicases function throughout biology to manipulate RNA structures. Over the past 12 years, we have used biochemical and biophysical approaches to delineate how DEAD-box helicases use local RNA unwinding to promote folding and structural rearrangements of RNAs with secondary and tertiary structure, and how a helicase can function as a general RNA chaperone. The proposed work delves further into how conformational changes in the helicase core produce ATP-dependent local RNA unwinding, a process that remains poorly understood and is a general requirement for DEAD-box helicases. We will also explore biological interactions and partners of Mss116, a S. cerevisiae protein that functions as a general RNA chaperone in mitochondria. (2) The DEAH-box family helicase DHX36 is an essential protein that binds specifically to RNA and DNA G-quadruplex structures (G4s) and uses ATP to unfold them. Our recent published work has defined key elements of the mechanism of G4 disruption. The proposed work addresses a key question ?how does the N-terminal domain of DHX36, which binds specifically to G4s, assist in their disruption? and tests the hypothesis that DHX36 functions as a chaperone for G4s in telomerase RNA. (3) CRISPR-Cas nucleases have enormous potential for gene editing applications and beyond, but our knowledge of the molecular steps of RNA assembly, DNA targeting, and DNA cleavage are at the very early stages. We recently applied quantitative kinetics approaches to investigate how the Cas12a nuclease is able to achieve higher specificity for target DNA than the benchmark Cas9. Our proposed work builds on this understanding by probing the physical origin of the high specificity and how protein rearrangements are linked to target DNA recognition. In addition, we will dissect the reaction steps and specificity determinants for pre- crRNA assembly with Cas12a, which have not been explored systematically. In each research area, we strive to answer basic research questions that are likely to give important and generalizable insights. Our work also has implications for understanding and treating diseases, as defects in these proteins are linked to many diseases including cancer, and CRISPR-Cas enzymes have emerged as key tools to combat genetic diseases.